Campus Units

Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of, Chemistry

Document Type

Article

Publication Version

Accepted Manuscript

Publication Date

3-2020

Journal or Book Title

Protein Expression and Purification

Volume

167

First Page

105540

DOI

10.1016/j.pep.2019.105540

Abstract

Various fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partner and is promptly removed upon digestion with a protease. The presented protocol allows for the production of mg amounts of Alkbh5 and FTO in 1L of both rich and minimal media. We developed a liquid chromatography-mass spectrometry (LC-MS)-based assay to confirm that both proteins are enzymatically active. Furthermore, the LC-MS method developed here is applicable to other members of the AlkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases. The superior protein yield, afforded by our expression and purification method, will facilitate biochemical investigations into the biological function of the human RNA demethylases and endorse employment of EIN as a broadly applicable fusion tag for recombinant expression projects.

Comments

This is a manuscript of an article published as Khatiwada, Balabhadra, Jeffrey A. Purslow, Eric S. Underbakke, and Vincenzo Venditti. "N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5." Protein Expression and Purification (2019): 105540. DOI: 10.1016/j.pep.2019.105540. Posted with permission.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Copyright Owner

Elsevier Inc.

Language

en

File Format

application/pdf

Available for download on Sunday, November 15, 2020

Published Version

Included in

Chemistry Commons

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